A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose

Recycle batch fermentations using immobilized cells of Propionibacterium acidipropionici were studied for propionate production from whey permeate, de-lactose whey permeate, and acid whey. Cells were immobilized in a spirally wound fibrous sheet packed in a 0.5-L column reactor, which was connected to a 5-L stirred tank batch fermentor with recirculation. The immobilized cells bioreactor served as a breeder for these recycle batch fermentations. High fermentation rates and conversions were obtained with these whey media without nutrient supplementation. It took approximately 55 h to ferment whey permeate containing approximately 45 g/L lactose to approximately 20 g/L propionic acid. Higher propionate concentrations can be produced with various concentrated whey media containing more lactose. The highest propionic acid concentration obtained with the recycle batch reactor was 65 g/L, which is much higher than the normal maximum concentration of 35 to 45 g/L reported in the literature. The volumetric productivity ranged from 0.22 g/L . h to 0.47 g/L . h, depending on the propionate concentration and whey medium used. The corresponding specific cell productivity was 0.033 to 0.07 g/L . g cell. The productivity increased to 0.68 g/L . h when whey permeate was supplemented with 1% (w/v) yeast extract. Compared with conventional batch fermentation, the recycle batch fermentation with the immobilized cell bioreactor allows faster fermentation, produces a higher concentration of product, and can be run continually without significant downtime. The process also produced similar fermentation results with nonsterile whey media. (c) 1995 John Wiley & Sons, Inc.     

Enhanced poly(L-malic acid) production from pretreated cane molasses by Aureobasidium pullulans in fed-batch fermentation

Poly(L-malic acid) (PMA) is a natural polyester with many attractive properties for biomedical application. However, the cost of PMA production is high when glucose is used as a carbon source. To solve this problem, cane molasses as a low-cost feedstock was applied for the production of PMA. Six pretreatment methods were applied to cane molasses before fermentation. Pretreatment with combined tricalcium phosphate, potassium ferrocyanide, and sulfuric acid (TPFSA) removed significant amounts of metal ions from cane molasses. The PMA concentration increased from 5.4?g/L (untreated molasses) to 36.9?g/L (TPFSA-pretreated molasses) after fermentation in shake flasks. A fed-batch fermentation strategy was then developed. In this method, TPFSA-pretreated cane molasses solution was continuously fed into the fermentor to maintain the total sugar concentration at 20?g/L. This technique generated approximately 95.4?g/L PMA with a productivity of 0.57?g/L/hr. The present study indicated that fed-batch fermentation using pretreated cane molasses is a feasible technique for producing high amounts of PMA.     

Control of feed rate to a fed-batch culture using a heat-flux sensor

Summary Substrate inhibition in batch fermentations can be avoided by employing the fed-batch technique in which substrate concentration is kept at low levels by a programmed feed rate. This research demonstrates the use of a heat-flux sensor to control substrate addition by continuously monitoring evolving heat which is proportional to fermentation rate. Batch fermentation with 240 g/L glucose in the medium was compared with a fed-batch starting with 20 g/L glucose in the medium and increased, with 500 g/L glucose, to a final equivalent glucose concentration of 240 g/L. The batch fermentation produced 106 g/L ethanol in 39 hr at 2.72 g/L/h, while the best fed-batch produced 114 g/L ethanol in 34 hr at 3.35 g/L/h with the same nutrients.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Fed-batch sorbose fermentation using pulse and multiple feeding strategies for productivity improvement

Microbial oxidation of D-sorbitol tol-sorbose byAcetobacter suboxydans is of commercial importance since it is the only biochemical process in vitamin C synthesis. The main bottleneck in the batch oxidation of sorbitol to sorbose is that the process is severely inhibited by sorbitol. Suitable fed-batch fermentation designs can eliminate the inherent substrate inhibition and improve sorbose productivity. Fed-batch sorbose fermentations were conducted by using two nutrient feeding strategies. For fed-batch fermentation with pulse feeding highly concentrated sorbitol (600 g/L) along with other nutrients were fed intermittently in four pulses of 0.5 liter in response to the increased DO signal. The fed-batch fermentation was over in 24 h with a sorbose productivity of 13.40 g/L/h and a final sorbose concentration of 320.48 g/L. On the other hand, in fed-batch fermentation with multiple feeds, two pulse feeds of 0.5 liter nutrient medium containing 600 g/L sorbitol was followed by the addition of 1.5 liter nutrient medium containing 600 g/L sorbitol at a constant feed rate of 0.36 L/h till the full working capacity of the reactor. The fermentation was completed in 24 h with an enhanced sorbose productivity of 15.09 g/L/h and a sorbose concentration of 332.60 g/L. The sorbose concentration and productivity obtained by multiple feeding of nutrients was found to be higher than that obtained by pulse feeding and was therefore a better strategy for fed-batch sorbose fermentation.     

Production of ethanol from molasses at 45 °C using alginate-immobilized Kluyveromyces marxianus imb3

The thermotolerant, ethanol-producing yeast strain, Kluyveromyces marxianus IMB3, has been immobilized in calcium alginate matrices. The ability of the biocatalyst to produce ethanol from cane molasses originating in Guatemala, Honduras, Senegal, Guyana and the Philippines was examined. In each case the molasses was diluted to yield a sugar concentration of 140?g/l and fermentations were carried out in batch-fed mode at 45?°C. During the first 24 hours, the maximum ethanol concentrations obtained ranged from 43–57?g/l with optimum production on the molasses from Honduras. Ethanol production during subsequent re-feeding of the fermentations at 24-hour intervals over a 120-hour period, decreased steadily to concentrations ranging from 20–36?g/l and it was found that ethanol productivity remained highest in fermentations containing the molasses from Guyana. When each set of fermentations was re-fed at 120?h and allowed to continue for 48?h, ethanol production again increased to a maximum with concentrations ranging from 25–52?g/l. It was also found however, that increasing the time between re-feeding at this stage in fermentation had a detrimental effect on the functionality of the biocatalyst.     

Culture medium containing glucose and glycerol as a mixed carbon source improves ε-poly-<Emphasis Type="SmallCaps">l</Emphasis>-lysine production by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. M-Z18

To improve the efficiency of ε-poly-l-lysine (ε-PL) production by Streptomyces sp. M-Z18, batch and fed-batch fermentations with glucose and glycerol (co-fermentations) were performed. The batch fermentations showed that the initial ratio of glucose to glycerol plays an important role in glucose/glycerol co-fermentation. The optimal glucose/glycerol weight ratio was 30/30; this resulted in a maximum ε-PL productivity of 5.26 g/L/d. Glucose and glycerol were consumed synergistically during the co-fermentation process, and the length of time during which the substrate was exhausted was significantly shortened compared with the single carbon source fermentation. Under optimized conditions, fed-batch fermentations with glucose and glycerol as a mixed carbon source achieved maximum ε-PL concentration and productivity values of 35.14 g/L and 4.85 g/L/d, respectively. These values were respectively 1.43- and 1.39-, and 1.17- and 1.16-folds higher than those obtained from fermentations with glucose and glycerol as single carbon sources. The present study is the first to suggest that glucose/glycerol co-fermentation may be an efficient strategy for ε-PL production by Streptomyces sp. M-Z18.     

Ergosterol production from molasses by genetically modified <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ergosterol is an economically important metabolite produced by fungi. Recombinant Saccharomyces cerevisiae YEH56(pHXA42) with increased capacity of ergosterol formation was constructed by combined overexpression of sterol C-24(28) reductase and sterol acyltransferase in the yeast strain YEH56. The production of ergosterol by this recombinant strain using cane molasses (CM) as an inexpensive carbon source was investigated. An ergosterol content of 52.6 mg/g was obtained with 6.1 g/l of biomass from CM medium containing 60 g/l of total sugar in 30 h in shake flask. The ergosterol yield was enhanced through the increasing cell biomass by supplementation of urea to a concentration of 6 g/l in molasses medium. Fermentation was performed in 5-l bioreactor using the optimized molasses medium. In batch fermentation, the effect of agitation velocity on ergosterol production was examined. The highest ergosterol yield was obtained at 400 rpm that increased 60.4 mg/l in comparison with the shake flask culture. In fed-batch fermentation, yeast cells were cultivated, firstly, in the starting medium containing molasses with 20 g/l of total sugar, 1.68 g/l of phosphate acid, and 6 g/l of urea (pH 5.4) for 5 h, then molasses containing 350 g/l of total sugar was fed exponentially into the bioreactor to keep the ethanol level in the broth below 0.5%. After 40 h of cultivation, the ergosterol yield reached 1,707 mg/l, which was 3.1-fold of that in the batch fermentation.     

Improvement of mannitol production by Lactobacillus brevis mutant 3-A5 based on dual-stage pH control and fed-batch fermentations

Lactobacillus brevis 3-A5 was isolated and expected to produce mannitol efficiently by regulating pH in batch and fed-batch fermentations. In 48 h batch fermentations with free and constant pH, the optimal pH for cell growth and mannitol production in the first 24 h of incubation was 5.5, whereas that for mannitol production in the second 24 h of incubation was 4.5. To achieve high cell density and mannitol yield simultaneously, a dual-stage pH control strategy was proposed based on the kinetic analysis of mannitol production. The pH value was controlled at 5.5 for the first 12 h of fermentation and subsequently shifted to 4.5 until the fermentation was completed. Under dual-stage pH control fermentation, a 103 g/L yield of mannitol with a volumetric production rate of 3.7 g/L/h was achieved after 28 h. The dual-stage pH control fed-batch fermentation strategy was further developed to improve mannitol yield, wherein the yield increased by 109 % to 215 g/L after 98 h of fermentation. This value is the highest yield of mannitol ever reported using L. brevis.     

Performance of batch, fed-batch, and continuous A-B-E fermentation with pH-control

Batch, fed-batch, and continuous A-B-E fermentations were conducted and compared with pH controlled at 4.5, the optimal range for solvent production. While the batch mode provides the highest solvent yield, the continuous mode was preferred in terms of butanol yield and productivity. The highest butanol yield and productivity found in the continuous fermentation at dilution rate of 0.1 h⁻¹ were 0.21 g-butanol/g-glucose and 0.81 g/L/h, respectively. In the continuous and fed-batch fermentation, the time needed for passing acidogenesis to solventogenesis was an intrinsic hindrance to higher butanol productivity. Therefore, a low dilution rate is suggested for the continuous A-B-E fermentation, while the fed-batch mode is not suggested for solvent production. While 3:6:1 ratio of acetone, butanol, and ethanol is commonly observed from A-B-E batch fermentation by Clostridium acetobutylicum when the pH is uncontrolled, up to 94% of the produced solvent was butanol in the chemostat with pH controlled at 4.5.     

Pullulan from peat hydrolyzate fermentation kinetics

The Luedeking-Piret equation was used to fit the kinetic data of pullulan fermentations from peat hydrolyzate substrate. In batch mode, the kinetic parameters m, n, alpha, and beta varied as a function of fermentation conditions: aeration rate, agitation speed, and temperature. In constant-feed fed-batch mode, the parameters Varied according to the feed rates. In peat hydrolyzate medium, the polysaccharide synthesis was strongly growth associated in batch and continuous fermentations but entirely growth associated in fedbatch fermentations. The fed-batch mode of fermentation with an appropriate feed rate is more advantageous with respect to batch and continuous fermentations. Therefore, if the fermentation is started batchwise and then followed by fed-batch mode at a constant feed rate, the overall polysaccharide productivity (g pullulan/L h) is significantly higher than those obtained with batch or continuous fermentations using the same total medium volume.     

Acetate production from whey lactose using co-immobilized cells of homolactic and homoacetic bacteria in a fibrous-bed bioreactor

Acetate was produced from whey lactose in batch and fed-batch fermentations using co-immobilized cells of Clostridium formicoaceticum and Lactococcus lactis. The cells were immobilized in a spirally wound fibrous sheet packed in a 0.45-L column reactor, with liquid circulated through a 5-L stirred-tank fermentor. Industrial-grade nitrogen sources, including corn steep liquor, casein hydrolysate, and yeast hydrolysate, were studied as inexpensive nutrient supplements to whey permeate and acid whey. Supplementation with either 2.5% (v/v) corn steep liquor or 1.5 g/L casein hydrolysate was adequate for the cocultured fermentation. The overall acetic acid yield from lactose was 0.9 g/g, and the productivity was 0.25 g/(L h). Both lactate and acetate at high concentrations inhibited the homoacetic fermentation. To overcome these inhibitions, fed-batch fermentations were used to keep lactate concentration low and to adapt cells to high-concentration acetate. The final acetate concentration obtained in the fed-batch fermentation was 75 g/L, which was the highest acetate concentration ever produced by C. formicoaceticum. Even at this high acetate concentration, the overall productivity was 0.18 g/(L h) based on the total medium volume and 1.23 g/(L h) based on the fibrous-bed reactor volume. The cells isolated from the fibrous-bed bioreactor at the end of this study were more tolerant to acetic acid than the original culture used to seed the bioreactor, indicating that adaptation and natural selection of acetate-tolerant strains occurred. This cocultured fermentation process could be used to produce a low-cost acetate deicer from whey permeate and acid whey.     

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 was investigated in semi-aerobic, pH-controlled, batch and fed-batch fermentations using a complex medium containing sucrose as the main source of carbon. The effects of sucrose concentration were studied in fed-batch fermentations in which a sucrose solution was added at stable feeding rates (5, 7, 9 and 10 g/l/h). The results showed that pediocin is produced as a product of the primary metabolism and its titer could be greatly improved by adjusting the sucrose feeding rate in fed-batch fermentation. The maximum titer of pediocin of 145 AU/ml was obtained in the fed-batch culture with 7 g/l/h feeding rate and that was 119% higher compared to the titer obtained in batch culture. Higher feeding rates (9 and 10 g/l/h) resulted in decreased pediocin yields while biomass levels appeared to be rather unaffected. The specific rate of pediocin formation was also sensitive to sucrose concentration levels. A mathematical model developed on the basis of well-known rate equations for batch and fed-batch cultures and growth associated production, described successfully cell growth, sucrose assimilation, lactate production and pediocin production in fed-batch culture.     

甘蔗渣和糖蜜混合发酵制备燃料丁醇

以抗逆突变株Clostridium beijerinckii IB4为研究对象,葡萄糖为C源,对其进行补料分批发酵过程的优化,同时将该优化工艺应用于甘蔗渣和糖蜜混合发酵制备燃料丁醇。结果表明:在5 L发酵罐中,先加入作为还原糖的甘蔗渣酸解糖液10 g/L,16 h后补加甘蔗糖蜜30 g/L,于35℃、100 r/min发酵50 h,丁醇和总溶剂产量分别达到11.1和15.3 g/L,丁醇比例高达72.5%。     

The acetone butanol fermentation on glucose and xylose. II. Regulation and kinetics in fed-batch cultures

The kinetics in fed-batch cultures of acetone butanol fermentation by Clostridium acetobutylicum is compared on glucose, xylose, and mixtures of both sugars. The final conversion yield of sugars into solvents always increases with the sugar feeding rate. At low feeding rates, the sugar concentration in the medium becomes limiting, which results in a slower cellular growth, a slower metabolic transition from an acid to a solvent fermentation and, thus, a higher accumulation of acids. It is only at sufficiently high feeding rates that fed-batch fermentations yield kinetic results comparable to those of batch fermentations. With mixtures of glucose and xylose, because of a maintained low glucose level, both sugars are taken up at the same rate during a first fermentation period. An earlier accumulation of xylose when the fermentation becomes inhibited suggest that xylose utilization is inhibited when the catabolic flux of glucose alone can satisfy the metabolic activity of the cell. Kinetic results with batch and fed-batch fermentations indicate several important features of the regulation of C. acetobutylicum metabolism: an early inhibition by the produced acids; an initiation of solvent formation between 4 and 6 g/L acetic and butyric acid depending on the metabolic activity of the cell; a metabolic transition from acids to solvents production at a rate closely related to the rate of sugar uptake; during solvent production, a reassimilation of acids above a minimal rate of sugar consumption of 0.2 h(-1); a final inhibition of the fermentation at a total butanol and acids concentration of ca. 20 g/L.     

Optimisation of L-lysine production byCorynebacterium sp in fed-batch cultures

Summary The influence of initial concentration of glucose from 60 to 233 g/l on the production of L-lysine byCorynebacterium sp was studied first in batch culture. The maximum conversion rate into L-lysine was obtained at 165 g/l and the best specific production rate for L-lysine was observed at 65 g/l of glucose. In fed-batch fermentations, better conversion and the specific production rates were obtained. Maintaining of a high glucose concentration in the fed-batch technique allowed a 54% increase of the L-lysine production compared to the batch culture.     

Enhanced 2,3-butanediol production in fed-batch cultures of free and immobilized Bacillus licheniformis DSM 8785

2,3-Butanediol (2,3-BD) is a valuable bulk chemical with particular use in industry. 2,3-BD has a potential as solvent and fuel additive, as carrier for pharmaceuticals, or as feedstock for the production of synthetic rubber. Until now, the highest 2,3-BD concentrations were obtained with risk group 2 microorganisms (e.g., Klebsiella oxytoca). In this study, the nonpathogenic bacterium Bacillus licheniformis DSM 8785 was used for 2,3-BD production from glucose. In batch experiments, a maximum 2,3-BD concentration of 72.6 g/L was reached from 180 g/L glucose after 86 h. The yield was 0.42 g/g glucose and the productivity was 0.86 g/(L h). During fed-batch cultivation, 2,3-BD production could be increased up to 144.7 g/L, with a productivity of 1.14 g/(L h). Additionally, repeated batch/fed-batch experiments were conducted using immobilized B. licheniformis in the form of LentiKats®. Results showed a high activity and stability of the immobilizates even after multiple medium replacements, as well as 2,3-BD concentrations, yields, and productivities similar to those obtained with free cells. To our knowledge, these results show the highest 2,3-BD concentration reported so far using a risk group 1 microorganism in general and B. licheniformis in particular. Furthermore, productivity lies in the same range with data reported from risk group 2 strains, which makes B. licheniformis DSM 8785 a suitable candidate for large-scale fermentation processes.     

Utilization of Molasses Sugar for Lactic Acid Production by Lactobacillus delbrueckii subsp. delbrueckii Mutant Uc-3 in Batch Fermentation

Efficient lactic acid production from cane sugar molasses by Lactobacillus delbrueckii mutant Uc-3 in batch fermentation process is demonstrated. Lactic acid fermentation using molasses was not significantly affected by yeast extract concentrations. The final lactic acid concentration increased with increases of molasses sugar concentrations up to 190 g/liter. The maximum lactic acid concentration of 166 g/liter was obtained at a molasses sugar concentration of 190 g/liter with a productivity of 4.15 g/liter/h. Such a high concentration of lactic acid with high productivity from molasses has not been reported previously, and hence mutant Uc-3 could be a potential candidate for economical production of lactic acid from molasses at a commercial scale.     

Lactobacillus reuteri CRL 1101 highly produces mannitol from sugarcane molasses as carbon source

Mannitol is a natural polyol extensively used in the food industry as low-calorie sugar being applicable for diabetic food products. We aimed to evaluate mannitol production by Lactobacillus reuteri CRL 1101 using sugarcane molasses as low-cost energy source. Mannitol formation was studied in free-pH batch cultures using 3-10% (w/v) molasses concentrations at 37?°C and 30?°C under static and agitated conditions during 48?h. L. reuteri CRL 1101 grew well in all assayed media and heterofermentatively converted glucose into lactic and acetic acids and ethanol. Fructose was used as an alternative electron acceptor and reduced it to mannitol in all media assayed. Maximum mannitol concentrations of 177.7?±?26.6 and 184.5?±?22.5?mM were found using 7.5% and 10% molasses, respectively, at 37?°C after 24-h incubation. Increasing the molasses concentration from 7.5% up to 10% (w/v) and the fermentation period up to 48?h did not significantly improve mannitol production. In agitated cultures, high mannitol values (144.8?±?39.7?mM) were attained at 8?h of fermentation as compared to static ones (5.6?±?2.9?mM), the highest mannitol concentration value (211.3?±?15.5?mM) being found after 24?h. Mannitol 2-dehydrogenase (MDH) activity was measured during growth in all fermentations assayed; the highest MDH values were obtained during the log growth phase, and no correlation between MDH activities and mannitol production was observed in the fermentations performed. L. reuteri CRL 1101 successfully produced mannitol from sugarcane molasses being a promising candidate for microbial mannitol synthesis using low-cost substrate.     

Enhanced 2,3-butanediol production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> SDM

Enhanced 2,3-butanediol (BD) production was carried out by Klebsiella pneumoniae SDM. The nutritional requirements for BD production by K. pneumoniae SDM were optimized statistically in shake flask fermentations. Corn steep liquor powder and (NH₄)₂HPO₄ were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform fed-batch fermentations with K. pneumoniae SDM. BD production was then studied in a 5-l bioreactor applying different fed-batch strategies, including pulse fed batch, constant feed rate fed batch, constant residual glucose concentration fed batch, and exponential fed batch. The maximum BD concentration of 150 g/l at 38 h with a diol productivity of 4.21 g/l h was obtained by the constant residual glucose concentration feeding strategy. To the best of our knowledge, these results were new records on BD fermentation. Cuiqing Ma and Ailong Wang contributed equally to this work.     

Optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae AS-22 in batch, fed-batch, and continuous cultures

Production of a novel cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae AS-22 strain, which converts starch predominantly to alpha-CD at high conversion yields, in batch, fed-batch, and continuous cultures, is presented. In batch fermentations, optimization of different operating parameters such as temperature, pH, agitation speed, and carbon-source concentration resulted in more than 6-fold increase in CGTase activity. The enzyme production was further improved by two fed-batch approaches. First, using glucose-based feed to increase cell density, followed by starch-based feed to induce enzyme production, resulted in high cell density of 76 g dry cell weight/L, although the CGTase production was low. Using the second approach of a single dextrin-based feed, 20-fold higher CGTase was produced compared to that in batch fermentations with media containing tapioca starch. In continuous operation, more than 8-fold increase in volumetric CGTase productivity was obtained using dextrin-based media compared to that in batch culture using starch-based media.